Early analysis and the possibility of directly genotyping the pathogenic agent are among the principal objectives of research in the diagnostic field. The development of new diagnostic assays should also take into account a degree of compatibility to high-throughput screening methods and a high level of sensitivity for diagnoses made as soon as possible after the occurrence of the infection. In developing countries it is also important to take into account the ease of handling of biological samples, for more widespread distribution of the diagnostic assays.
There are currently three types of in vitro diagnostic systems: direct culture of the pathogenic agent from the biological sample, which is the so-called “gold standard” of diagnostic assays; immunological assays based on the detection of products or antigens of the infectious agent; and indirect immunological assays that can detect antibodies produced against the infectious agent during or after infection.
In the first system, the principal disadvantage is that the biological sample must be considered to be at risk, inasmuch as it can potentially transmit the pathogenic agent, whereas in the indirect detection of the antibodies there is no possibility of discriminating between past and current infections.
Molecular diagnostic methods have been developed that are based on the detection of the nucleic acids of the pathogenic agent in the blood or plasma samples, or in the cell cultures, taken from the patient. These tests are usually much more sensitive than the immunoassays. For this reason, and because of their specificity, they are extremely promising, but usually require special equipment and qualified personnel.
Molecular diagnostic methods based on transrenal DNA (TrDNA) have been described and their utility for monitoring the fate of allogeneic transplants, to detect the sex of a fetus, and to screen the presence of tumor markers was demonstrated. In particular, U.S. Pat. No. 6,251,638 describes an analytical method for detecting male fetal DNA in the urine of pregnant women; in U.S. Pat. No. 6,287,820, the invention is aimed at the diagnosis of tumors, particularly of adenocarcinomas (of the colon and pancreas); and in U.S. Pat. No. 6,492,144, the transrenal nucleic-acid analysis method is used to monitor the progress of allogeneic transplants, using known methods for molecular analysis. The presence of identifiable transrenal DNA in urine, in the fraction of DNA fragments consisting of 150 base pairs or more, was shown (Al-Yatama et al. (2001), “Detection of Y-chromosome-specific DNA in the plasma and urine of pregnant women using nested polymerase chain reaction”; Prenat Diagn, 21:399-402; and Utting, M., et al. (2002), “Microsatellite analysis of free tumor DNA in urine, serum, and plasma of patients: A minimally invasive method for the detection of bladder cancer”; Clin Cancer Res, 8:35-40).
Molecular detection of TrDNA in urine is performed using techniques that are very well known in the art and widely used in laboratory practice, such as PCR (polymerase chain reaction), hybridization, or the so-called “cycling probe reaction.”
The presence of transrenal DNA has been explained as being the result of phenomenon of apoptosis. In the process of apoptosis or programmed cell death the nuclear DNA is cleaved into nucleosomes and oligomers, which subsequently, as a part of apoptotic process, are phagocytozed and removed from the organism. (Umansky, S. R., et al. (1982), “In vivo DNA degradation in thymocytes of gamma-irradiated or hydrocortisone-treated rats”; Biochim. Biophys. Acta, 655:9-17). A portion of this degraded DNA, though, escapes the phagocytosis, and appears in the bloodstream (Lichtenstein, A. V., et al. (2001), “Circulating nucleic acids and apoptosis”; Ann N.Y. Acad Sci, 945:239-249), and, as confirmed in the above-referred patents, also in urine.
The presence of viral DNA that originates from sources outside of the urinary tract, in urine has not been described until now. Meanwhile, circulation of viral DNA released from the genome of transfected cell in the plasma has been shown: for example, fragments of Epstein-Barr viral DNA were detected in plasma of patients with nasopharyngeal carcinoma (Chan, K. C., et al. (2002), “Molecular characterization of circulating EBV DNA in the plasma of nasopharyngeal carcinoma and lymphoma patients”; Cancer Res 63:2028-2032), and in the case of human papilloma virus (HPV) in the plasma of patients with cervical cancer (Pornthanakasem, W., et al. (2001), “Human Papillomavirus DNA in plasma of patients with cervical cancer”; BMC Cancer 1:2).